Cycloalkyne derivatized saccharides

ABSTRACT

This disclosure provides novel saccharide derivatives, conjugates, and methods for making the derivatives and conjugates.

TECHNICAL FIELD

This invention is in the field of saccharide derivatives, conjugatesincluding saccharides and methods for producing the saccharidederivatives and conjugates. The conjugates are useful for immunisation.

BACKGROUND OF THE INVENTION

The capsular saccharides of bacteria have been used for many years invaccines against capsulated bacteria. As saccharides are T-independentantigens, however, they are poorly immunogenic. Conjugation to a carriercan convert T-independent antigens into T-dependent antigens, therebyenhancing memory responses and allowing protective immunity to develop.

While classical procedures for conjugation (reductive amination, amidebond formation, etc.) rely on the random reaction of the polysaccharideto the amines of the carrier protein, novel conjugation methods enablingsite specific installation of a ligand onto a protein are emerging [1].Site specific conjugation, besides providing more homogeneousbiomolecules as vaccine candidates, could aid to preserve theimmunogenicity of the protein.

The click chemistry approach has been described as a method for theformation of complex substances by joining small subunits together in amodular fashion[2, 3]. Various forms of click chemistry reaction areknown in the art, such as the Huisgen 1,3-dipolar cycloaddition coppercatalyzed reaction[4], which is often referred to as the “clickreaction”. Other alternatives include cycloaddition reactions such asthe Diels-Alder, nucleophilic substitution reactions (especially tosmall strained rings like epoxy and aziridine compounds), carbonylchemistry formation of urea compounds and reactions involvingcarbon-carbon double bonds, such as alkynes in thiol-yne reactions.

The azide-alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-di-substituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often not required[5]. The azide and alkyne functional groups are largely inert towardsbiomolecules in aqueous medium, allowing the reaction to occur incomplex solutions. The triazole formed is chemically stable and is notsubject to enzymatic cleavage, making the click chemistry product highlystable in biological systems. However, the copper catalyst is toxic toliving cells, precluding biological applications.

A copper-free click reaction has been proposed [6], which uses ringstrain (in a cyclooctyne ring) in place of the copper catalyst topromote a [3+2] azide-alkyne cycloaddition reaction. The closed ringstructure induces a substantial bond angle deformation of the acetylene,which is highly reactive with azide groups to form a triazole.

It is an object of the present invention to provide further and improvedmethods for derivatizing saccharides. It is another object of thepresent invention to provide further and improved methods forconjugating saccharides to various moieties, such as carrier proteins.Is is also an objection of the invention to provide a conjugation methodwhich yields conjugates with more uniform structures. It is also anobject of the invention to provide conjugates with improved immunogenicproperties.

SUMMARY OF THE INVENTION

The inventors have developed new processes for derivatizing saccharidesand for conjugation of such saccharide derivatives to other moieties.The inventors have also produced novel saccharide derivatives andconjugates which have improved properties over saccharide derivativesand conjugates known in the art. In particular, the conjugates of theinvention may have improved immunological properties.

In one aspect, the invention provides a method of derivatizing asaccharide comprising attaching an eight-membered cycloalkyne group tothe saccharide. The invention also provides a saccharide derivativecomprising an eight-membered cycloalkyne group. The saccharidederivative comprising an eight-membered cycloalkyne group may beobtained or obtainable by the method of derivatizing a saccharide.

In another aspect, the invention provides a method of conjugating asaccharide derivative to an azide-containing moiety, comprising reactingthe eight-membered cycloalkyne group with the azide to form a triazolelinkage. The invention also provides a conjugate of a saccharidederivative and an azide-containing moiety, wherein the conjugate has theformula R—S-T, wherein R comprises a residue of the saccharidederivative, S is a triazole group fused to an eight-membered cycloalkylgroup and T comprises a residue of the moiety azide-containing moiety.

The conjugate may be obtained or obtainable by the method of conjugatinga saccharide derivative to an azide-containing moiety of the invention.

The present invention also relates to pharmaceutical compositionscomprising a conjugate of the invention in combination with apharmaceutically acceptable carrier.

The present invention further relates to methods for raising an immuneresponse in a mammal, comprising administering a conjugate orpharmaceutical composition of the invention to the mammal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows three cyclooctyne-containing compounds.

FIG. 2 shows a general reaction scheme for the attachment of acyclooctyne group to a GBS serotype II saccharide.

FIG. 3 shows the structure of GBS serotype V saccharide with cyclooctynegroup attached (I).

FIG. 4 shows the structure of GBS serotype II saccharide withcyclooctyne group attached (II).

FIG. 5 shows a general reaction scheme for the conjugation of saccharidederivative (II) to a GBS80 carrier protein via a tyrosine residue.

FIG. 6 shows the results of SDS-PAGE characterization for conjugate A(1=MW, 2=GBS80-Y—N₃, 3=GBS80-Y—N₃/PSV after purification).

FIG. 7 shows the results of SDS-PAGE characterization for conjugate B(1=MW, 2=GBS80-Y—N₃, 3=GBS80-Y—N₃/PSII after purification).

FIG. 8 shows the results of SDS-PAGE characterization for conjugate C(1=MW, 2=GBS67-Y—N₃, 3=GBS67-Y—N₃/PSII after purification,4=GBS67-Y—N₃/PSII after purification).

FIG. 9 shows the results of SDS-PAGE characterization for conjugate D(1=MW, 2=GBS67-Y—N₃, 3=GBS67-Y—N₃/PSII after purification,4=GBS67-Y—N₃/PSII after purification).

FIG. 10 shows the structure of MenY saccharide with cyclooctyne groupattached (III).

FIG. 11 shows the results of SDS-PAGE characterization for conjugate E(1=MW, 2=CRM₁₉₇-Y—N₃, 3=CRM₁₉₇-Y—N₃/MenY).

FIG. 12 shows ELISA immunoassay results for determination of IgG titersagainst GB S serotype II saccharide antigens (for 1.0 μg carbohydratedose).

FIG. 13 shows ELISA immunoassay results for determination of IgG titersagainst GB S serotype II saccharide antigens (for 0.5 μg carbohydratedose).

FIG. 14 shows ELISA immunoassay results for determination of IgG titersagainst GB S serotype II saccharide antigens (for 1.0 μg protein dose).

FIG. 15 shows opsonophagocytosis assay results using GBS strains.

FIG. 16 shows immune response of various antigens against GBS serotypeII saccharide.

FIG. 17 shows immune response of various antigens against GBS80.

FIG. 18 shows the structure of a construct prepared via tyrosineselective conjugation of a MenY dimer to CRM197.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves methods of derivatizing a saccharide and methodsof conjugating a saccharide derivative to an azide-containing moiety.The invention also involves novel saccharide derivatives and conjugates.The features of these methods, saccharide derivatives and conjugates aredescribed in detail below.

Method of Derivatizing a Saccharide

The invention is based on novel saccharide derivatives and methods ofproducing such saccharide derivatives.

The Saccharide

The saccharide used in the methods of the invention may be anysaccharide, particularly a saccharide from a pathogenic organism.Exemplary saccharides for use in the methods of the invention aredescribed below. In particular, the saccharide may be a bacterialsaccharide, e.g. a bacterial capsular saccharide.

The saccharides may be used in the form of oligosaccharides. These areconveniently formed by fragmentation of purified polysaccharide (e.g. byhydrolysis), which will usually be followed by purification of thefragments of the desired size. Saccharides may be purified from naturalsources. As an alternative to purification, saccharides may be obtainedby total or partial synthesis.

S. Agalactiae Capsular Saccharides

Preferred bacterial capsular saccharides include those fromStreptococcus agalactiae (“GBS”). The capsular saccharide is covalentlylinked to the peptidoglycan backbone of GBS, and is distinct from thegroup B antigen, which is another saccharide that is attached to thepeptidoglycan backbone.

GBS is a leading cause of severe bacterial infections in early 3 monthsof life among newborns and of septic morbidity among mothers [7]. GBS isalso an important cause of morbidity and mortality among non-pregnantadults, particularly among old people and adults with underlying medicalconditions. All GBS strains possess a capsular polysaccharide (CPS) ontheir surface, which is a major virulence factor. Ten different CPSserotypes have been characterized (Ia, Ib, II, III, IV, V, VI, VII, VIIIand IX), of which five (Ia, Ib, II, III, V) are responsible for themajority of the neonatal disease in North America and Europe. Monovalentconjugate vaccines have been prepared against serotypes Ia, Ib, II, III,IV, V, VI, VII, VIII and effectiveness demonstrated in animal models 3.Recently, it has been demonstrated that GBS pilus proteins, besidesbeing important structures in bacterial adhesion and invasion, seem tobe more conserved than those of other Gram-positive bacteria [8].

The GBS capsular saccharides are chemically related, but areantigenically very different. All GBS capsular saccharides share thefollowing trisaccharide core:

-   -   β-D-GlcpNAc(1→3)β-D-Galp(1→4)β-D-Glcp

The various GBS serotypes differ by the way in which this core ismodified.

GBS-related disease arises primarily from serotypes Ia, Ib, II, III, IV,V, VI, VII, and VIII, with over 85% being caused by five serotypes: Ia,Ib, III & V. The invention may use a saccharide from any serotype, inparticular serotypes Ia, Ib, II, III & V.

Saccharides used in the methods of the invention may be in their nativeform, or may have been modified. For example, the saccharide may beshorter than the native capsular saccharide, or may be chemicallymodified. In particular, the serotype V capsular saccharide used in theinvention may be modified as described in refs. 9 and 10. For example, aserotype V capsular saccharide that has been substantially desialylated.Desialylated GBS serotype V capsular saccharide may be prepared bytreating purified GBS serotype V capsular saccharide under mildly acidicconditions (e.g. 0.1M sulphuric acid at 80° C. for 60 minutes) or bytreatment with neuraminidase, as described in reference 9. Thesaccharide used according to the invention may be a substantiallyfull-length capsular polysaccharide, as found in nature, or it may beshorter than the natural length. Full-length polysaccharides may bedepolymerised to give shorter fragments for use with the invention e.g.by hydrolysis in mild acid, by heating, by sizing chromatography, etc.In particular, the serotype II and/or III capsular saccharides used inthe invention may be depolymerised as described in refs. 11 and 12.

The saccharide may be chemically modified relative to the capsularsaccharide as found in nature. For example, the saccharide may bede-O-acetylated (partially or fully), de-N-acetylated (partially orfully), N-propionated (partially or fully), etc. De-acetylation mayoccur before, during or after conjugation, but preferably occurs beforeconjugation. Depending on the particular saccharide, de-acetylation mayor may not affect immunogenicity. The relevance of O-acetylation on GBSsaccharides in various serotypes is discussed in reference 13, and insome embodiments O-acetylation of sialic acid residues at positions 7, 8and/or 9 is retained before, during and after conjugation e.g. byprotection/de-protection, by re-acetylation, etc. However, typically theGBS saccharide used in the present invention has substantially noO-acetylation of sialic acid residues at positions 7, 8 and/or 9. Inparticular, when the GBS saccharide has been purified by base extractionas described below, then O-acetylation is typically lost. The effect ofde-acetylation etc. can be assessed by routine assays.

Capsular saccharides can be purified by known techniques, as describedin 14. A typical process involves base extraction, centrifugation,filtration, RNase/DNase treatment, protease treatment, concentration,size exclusion chromatography, ultrafiltration, anion exchangechromatography, and further ultrafiltration. Treatment of GBS cells withthe enzyme mutanolysin, which cleaves the bacterial cell wall to freethe cell wall components, is also useful.

As an alternative, the purification process described in reference 15can be used. This involves base extraction, ethanol/CaCl₂ treatment,CTAB precipitation, and re-solubilisation. A further alternative processis described in reference 16.

N. meningitidis Capsular Saccharides

The saccharide may be a bacterial capsular saccharide. Exemplarybacterial capsular saccharides include those from N. meningitidis. Basedon the organism's capsular polysaccharide, various serogroups of N.meningitidis have been identified, including A, B, C, H, I, K, L, 29E,W135, X, Y & Z. The saccharide in the invention may be from any of theseserogroups. Typically, the saccharide is from one of the followingmeningococcal serogroups: A, C, W135 and Y.

The capsular saccharides will generally be used in the form ofoligosaccharides. These are conveniently formed by fragmentation ofpurified capsular polysaccharide (e.g. by hydrolysis), which willusually be followed by purification of the fragments of the desiredsize.

Fragmentation of polysaccharides is typically performed to give a finalaverage degree of polymerisation (DP) in the oligosaccharide of lessthan 30 (e.g. between 10 and 20, preferably around 10 for serogroup A;between 15 and 25 for serogroups W135 and Y, preferably around 15-20;between 12 and 22 for serogroup C; etc.). DP can conveniently bemeasured by ion exchange chromatography or by colorimetric assays [17].

If hydrolysis is performed, the hydrolysate will generally be sized inorder to remove short-length oligosaccharides [18]. This can be achievedin various ways, such as ultrafiltration followed by ion-exchangechromatography. Oligosaccharides with a degree of polymerisation of lessthan or equal to about 6 are preferably removed for serogroup A, andthose less than around 4 are preferably removed for serogroups W135 andY.

Chemical hydrolysis of saccharides generally involves treatment witheither acid or base under conditions that are standard in the art.Conditions for depolymerisation of capsular saccharides to theirconstituent monosaccharides are known in the art. One depolymerisationmethod involves the use of hydrogen peroxide [19]. Hydrogen peroxide isadded to a saccharide (e.g. to give a final H₂O₂ concentration of 1%),and the mixture is then incubated (e.g. at around 55° C.) until adesired chain length reduction has been achieved. The reduction overtime can be followed by removing samples from the mixture and thenmeasuring the (average) molecular size of saccharide in the sample.Depolymerization can then be stopped by rapid cooling once a desiredchain length has been reached

Serogroups C, W135 and Y

Techniques for preparing capsular polysaccharides from meningococci havebeen known for many years, and typically involve a process comprisingthe steps of polysaccharide precipitation (e.g. using a cationicdetergent), ethanol fractionation, cold phenol extraction (to removeprotein) and ultracentrifugation (to remove LPS) [e.g. see ref. 20].

A more preferred process [21] involves polysaccharide precipitationfollowed by solubilisation of the precipitated polysaccharide using alower alcohol. Precipitation can be achieved using a cationic detergentsuch as tetrabutylammonium and cetyltrimethylammonium salts (e.g. thebromide salts), or hexadimethrine bromide and myristyltrimethylammoniumsalts. Cetyltrimethylammonium bromide (‘CTAB’) is particularly preferred[22]. Solubilisation of the precipitated material can be achieved usinga lower alcohol such as methanol, propan-1-ol, propan-2-ol, butan-1-ol,butan-2-ol, 2-methyl-propan-1-ol, 2-methyl-propan-2-ol, diols, etc., butethanol is particularly suitable for solubilising CTAB-polysaccharidecomplexes. Ethanol may be added to the precipitated polysaccharide togive a final ethanol concentration (based on total content of ethanoland water) of between 50% and 95%.

After re-solubilisation, the polysaccharide may be further treated toremove contaminants. This is particularly important in situations whereeven minor contamination is not acceptable (e.g. for human vaccineproduction). This will typically involve one or more steps of filtratione.g. depth filtration, filtration through activated carbon may be used,size filtration and/or ultrafiltration.

Once filtered to remove contaminants, the polysaccharide may beprecipitated for further treatment and/or processing. This can beconveniently achieved by exchanging cations (e.g. by the addition ofcalcium or sodium salts).

Further and alternative methods for purification of meningococcalsaccharides are disclosed in references 19 & 23.

As an alternative to purification, capsular saccharides of the presentinvention may be obtained by total or partial synthesis e.g. Hibsynthesis is disclosed in ref. 24, and MenA synthesis in ref. 25.

The saccharide may be chemically modified e.g. it may be O-acetylated orde-O-acetylated. Any such de-O-acetylation or hyper-acetylation may beat specific positions in the saccharide. For instance, most serogroup Cstrains have O-acetyl groups at position C-7 and/or C-8 of the sialicacid residues, but about 15% of clinical isolates lack these O-acetylgroups [26,27]. The acetylation does not seem to affect protectiveefficacy (e.g. unlike the Menjugate™ product, the NeisVac-C™ productuses a de-O-acetylated saccharide, but both vaccines are effective). Theserogroup W135 saccharide is a polymer of sialic acid-galactosedisaccharide units. The serogroup Y saccharide is similar to theserogroup W135 saccharide, except that the disaccharide repeating unitincludes glucose instead of galactose. Like the serogroup C saccharides,the MenW135 and MenY saccharides have variable O-acetylation, but atsialic acid 7 and 9 positions [28].

Serogroup A

The method may include a serogroup A capsular saccharide antigen. Thesaccharide can be purified and conjugated in the same way as forserogroups C, W135 and Y (see above), although it is structurallydifferent whereas the capsules of serogroups C, W135 and Y are basedaround sialic acid (N-acetyl-neuraminic acid, NeuAc), the capsule ofserogroup A is based on N-acetyl-mannosamine, which is the naturalprecursor of sialic acid. The serogroup A saccharide is particularlysusceptible to hydrolysis, and its instability in aqueous media meansthat (a) the immunogenicity of liquid vaccines against serogroup Adeclines over time, and (b) quality control is more difficult, due torelease of saccharide hydrolysis products into the vaccine.

Native MenA capsular saccharide is a homopolymer of (α1→6)-linkedN-acetyl-D-mannosamine-1-phosphate, with partial O-acetylation at C3 andC4. The principal glycosidic bond is a 1-6 phosphodiester bond involvingthe hemiacetal group of C1 and the alcohol group of C6 of theD-mannosamine. The average chain length is 93 monomers. It has thefollowing formula:

A modified saccharide antigen has been prepared which retains theimmunogenic activity of the native serogroup A saccharide but which ismuch more stable in water. Hydroxyl groups attached at carbons 3 and 4of the monosaccharide units are replaced by a blocking group [refs. 29and 30].

The number of monosaccharide units having blocking groups in place ofhydroxyls can vary. For example, all or substantially all themonosaccharide units may have blocking groups. Alternatively, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the monosaccharideunits may have blocking groups. At least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29 or 30 monosaccharide units may have blocking groups.

Likewise, the number of blocking groups on a monosaccharide unit mayvary. For example, the number of blocking groups on any particularmonosaccharide unit may be 1 or 2.

Blocking groups to replace hydroxyl groups may be directly accessiblevia a derivatizing reaction of the hydroxyl group i.e. by replacing thehydrogen atom of the hydroxyl group with another group. Suitablederivatives of hydroxyl groups which act as blocking groups are, forexample, carbamates, sulfonates, carbonates, esters, ethers (e.g. silylethers or alkyl ethers) and acetals. Some specific examples of suchblocking groups are allyl, Aloc, benzyl, BOM, t-butyl, trityl, TBS,TBDPS, TES, TMS, TIPS, PMB, MEM, MOM, MTM, THP, etc. Other blockinggroups that are not directly accessible and which completely replace thehydroxyl group include C₁₋₁₂ alkyl, C₃₋₁₂ alkyl, C₅₋₁₂ aryl, C₅₋₁₂aryl-C₁₋₆ alkyl, NR¹R² (R¹ and R² are defined in the followingparagraph), H, F, Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃, CCl₃, etc.

Typical blocking groups are of the formula: —O—X—Y or —OR³ wherein: X isC(O), S(O) or SO₂; Y is C₁₋₁₂ alkyl, C₁₋₁₂ alkoxy, C₃₋₁₂ cycloalkyl,C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which may optionally besubstituted with 1, 2 or 3 groups independently selected from F, Cl, Br,CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃; or Y is NR¹R²; R¹ and R² areindependently selected from H, C₁₋₁₂ alkyl, C₃₋₁₂ cycloalkyl, C₅₋₁₂aryl, C₅₋₁₂ aryl-C₁₋₆ alkyl; or R¹ and R² may be joined to form a C₃₋₁₂saturated heterocyclic group; R³ is C₁₋₁₂ alkyl or C₃₋₁₂ cycloalkyl,each of which may optionally be substituted with 1, 2 or 3 groupsindependently selected from F, Cl, Br, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃;or R³ is C₅₋₁₂ aryl or C₅₋₁₂ aryl-C₁₋₆ alkyl, each of which mayoptionally be substituted with 1, 2, 3, 4 or 5 groups selected from F,Cl, Br, CO₂H, CO₂(C₁₋₆ alkyl), CN, CF₃ or CCl₃. When R³ is C₁₋₁₂ alkylor C₃₋₁₂ cycloalkyl, it is typically substituted with 1, 2 or 3 groupsas defined above. When R¹ and R² are joined to form a C₃₋₁₂ saturatedheterocyclic group, it is meant that R¹ and R² together with thenitrogen atom form a saturated heterocyclic group containing any numberof carbon atoms between 3 and 12 (e.g. C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀,C₁₁, C₁₂). The heterocyclic group may contain 1 or 2 heteroatoms (suchas N, O or S) other than the nitrogen atom. Examples of C₃₋₁₂ saturatedheterocyclic groups are pyrrolidinyl, piperidinyl, morpholinyl,piperazinyl, imidazolidinyl, azetidinyl and aziridinyl.

Blocking groups —O—X—Y and —OR³ can be prepared from —OH groups bystandard derivatizing procedures, such as reaction of the hydroxyl groupwith an acyl halide, alkyl halide, sulfonyl halide, etc. Hence, theoxygen atom in —O—X—Y is usually the oxygen atom of the hydroxyl group,while the —X—Y group in —O—X—Y usually replaces the hydrogen atom of thehydroxyl group.

Alternatively, the blocking groups may be accessible via a substitutionreaction, such as a Mitsonobu-type substitution. These and other methodsof preparing blocking groups from hydroxyl groups are well known.

Specific blocking groups for use in the invention are —OC(O)CF₃ [31] anda carbamate group OC(O)NR¹R², where R¹ and R² are independently selectedfrom C₁₋₆ alkyl. Typically, R¹ and R² are both methyl i.e. the blockinggroup is —OC(O)NMe₂. Carbamate blocking groups have a stabilizing effecton the glycosidic bond and may be prepared under mild conditions.

A particularly preferred blocking group is —OC(O)CH₃ [30]. Theproportion of 4- and/or 3-positions in the modified Neisseriameningitidis serogroup A saccharide that have this blocking group mayvary. For example, the proportion of 4-positions that have blockinggroups may be about 0%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or about 100%, with at least 80% and about 100% beingpreferred. Similarly, the proportion of 3-positions that have blockinggroups may be about 0%, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95% or about 100%, with at least 80% and about 100% beingpreferred. Typically, the proportion of 4- and 3-positions that haveblocking groups is about the same at each position. In other words, theratio of 4-positions that have blocking groups to 3-positions that haveblocking groups is about 1:1. However, in some embodiments, theproportion of 4-positions that have blocking groups may vary relative tothe proportion of 3-positions that have blocking groups. For example,the ratio of 4-positions that have blocking groups to 3-positions thathave blocking groups may be 1:20, 1:19, 1:18, 1:17, 1:16, 1:15, 1:14,1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or 1:2.Similarly, the ratio of 3-positions that have blocking groups to4-positions that have blocking groups may be 1:20, 1:19, 1:18, 1:17,1:16, 1:15, 1:14, 1:13, 1:12, 1:11, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,1:3 or 1:2.

Typical modified MenA saccharides contain n monosaccharide units, whereat least h % of the monosaccharide units do not have —OH groups at bothof positions 3 and 4. The value of h is 24 or more (e.g. 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 98, 99 or100) and is usually 50 or more. The absent —OH groups are blockinggroups as defined above.

Other typical modified MenA saccharides comprise monosaccharide units,wherein at least s of the monosaccharide units do not have —OH at the 3position and do not have —OH at the 4 position. The value of s is atleast 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90). The absent —OH groups are blocking groups as defined above.

Suitable modified MenA saccharides for use with the invention have theformula:

wherein

-   -   n is an integer from 1 to 100 (particularly an integer from 5 to        25, usually 15-25);    -   T is of the formula (A) or (B):

-   -   each Z group is independently selected from OH or a blocking        group as defined above; and    -   each Q group is independently selected from OH or a blocking        group as defined above;    -   Y is selected from OH or a blocking group as defined above;    -   E is H or a nitrogen protecting group;        and wherein more than about 7% (e.g. 8%, 9%, 10% or more) of the        Q groups are blocking groups. In some embodiments, the hydroxyl        group attached at carbon 1 in formula (A) is replaced by a        blocking group as defined above. In some embodiments, E in        formula (B) is the point of attachment to the cyclooctyne group.

Each of the n+2 Z groups may be the same or different from each other.Likewise, each of the n+2 Q groups may be the same or different fromeach other. All the Z groups may be OH. Alternatively, at least 10%, 20,30%, 40%, 50% or 60% of the Z groups may be OAc. Typically, about 70% ofthe Z groups are OAc, with the remainder of the Z groups being OH orblocking groups as defined above. At least about 7% of Q groups areblocking groups. Typically, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or even 100% of the Q groups are blocking groups.

Glucans

The saccharide may be a glucan. Glucans are glucose-containingpolysaccharides found inter alia in fungal cell walls. The α-glucansinclude one or more α-linkages between glucose subunits, whereasβ-glucans include one or more β-linkages between glucose subunits. Theglucan used in accordance with the invention includes p linkages, andmay contain only p linkages (i.e. no a linkages).

The glucan may comprise one or more β-1,3-linkages and/or one or moreβ-1,6-linkages. It may also comprise one or more β-1,2-linkages and/orβ-1,4-linkages, but normally its only p linkages will be β-1,3-linkagesand/or β-1,6-linkages.

The glucan may be branched or linear.

Full-length native β-glucans are insoluble and have a molecular weightin the megadalton range. It is preferred to use soluble glucans inconjugates of the invention. Solubilisation may be achieved byfragmenting long insoluble glucans. This may be achieved by hydrolysisor, more conveniently, by digestion with a glucanase (e.g. with aβ-1,3-glucanase or a β-1,6-glucanase). As an alternative, short glucanscan be prepared synthetically by joining monosaccharide building blocks.

Low molecular weight glucans are preferred, particularly those with amolecular weight of less than 100 kDa (e.g. less than 80, 70, 60, 50,40, 30, 25, 20, or 15 kDa). It is also possible to use oligosaccharidese.g. containing 60 or fewer (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51,50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4) glucose monosaccharide units.Within this range, oligosaccharides with between 10 and 50 or between 20and 40 monosaccharide units are preferred.

The glucan may be a fungal glucan. A ‘fungal glucan’ will generally beobtained from a fungus but, where a particular glucan structure is foundin both fungi and non-fungi (e.g. in bacteria, lower plants or algae)then the non-fungal organism may be used as an alternative source. Thusthe glucan may be derived from the cell wall of a Candida, such as C.albicans, or from Coccidioides immitis, Trichophyton verrucosum,Blastomyces dermatidis, Cryptococcus neoformans, Histoplasma capsulatum,Saccharomyces cerevisiae, Paracoccidioides brasiliensis, or Pythiumninsidiosum.

There are various sources of fungal β-glucans. For instance, pureβ-glucans are commercially available e.g. pustulan (Calbiochem) is aβ-1,6-glucan purified from Umbilicaria papullosa. β-glucans can bepurified from fungal cell walls in various ways. Reference 32, forinstance, discloses a two-step procedure for preparing a water-solubleβ-glucan extract from Candida, free from cell-wall mannan, involvingNaClO oxidation and DMSO extraction. The resulting product (‘Candidasoluble (3-D-glucan’ or ‘CSBG’) is mainly composed of a linearβ-1,3-glucan with a linear β-1,6-glucan moiety. Similarly, reference 33discloses the production of GG-zym from Calbicans. Such glucans from C.albicans, include (a) β-1,6-glucans with β-1,3-glucan lateral chains andan average degree of polymerisation of about 30, and (b) β-1,3-glucanswith β-1,6-glucan lateral chains and an average degree of polymerisationof about 4.

In some embodiments of the invention, the glucan is a β-1,3 glucan withsome β-1,6 branching, as seen in e.g. laminarins. Laminarins are foundin brown algae and seaweeds. The β(1-3):β(1-6) ratios of laminarins varybetween different sources e.g. it is as low as 3:2 in Eisenia bicyclislaminarin, but as high as 7:1 in Laminaria digititata laminarin [34].Thus the glucan used with the invention may have a β(1-3):β(1-6) ratioof between 1.5:1 and 7.5:1 e.g. about 2:1, 3:1, 4:1, 5:1, 6:1 or 7:1.Optionally, the glucan may have a terminal mannitol subunit, e.g. a1,1-α-linked mannitol residue [35]. The glucan may also comprise mannosesubunits.

In other embodiments, the glucan has exclusively or mainly β-1,3linkages, as seen in curdlan. These glucans may elicit better protectionthan glucans comprising other linkages, particularly glucans comprisingβ-1,3 linkages and a greater proportion of β-1,6 linkages. Thus theglucan may be made solely of β-1,3-linked glucose residues (e.g. linearβ-D-glucopyranoses with exclusively 1,3 linkages). Optionally, though,the glucan may include monosaccharide residues that are not β-1,3-linkedglucose residues e.g. it may include β-1,6-linked glucose residues. Theratio of β-1,3-linked glucose residues to these other residues should beat least 8:1 (e.g. ≥9:1, ≥10:1, ≥11:1, ≥12:1, ≥13:1, ≥14:1, ≥15:1,≥16:1, ≥17:1, ≥18:1, ≥19:1, ≥20:1, ≥25:1, ≥30:1, ≥35:1, ≥40:1, ≥45:1,≥50:1, ≥75:1, ≥100:1, etc.) and/or there are one or more (e.g. ≥1, ≥2,≥3, ≥4, ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, ≥12, etc.) sequences of at leastfive (e.g. ≥5, ≥6, ≥7, ≥8, ≥9, ≥10, ≥11, ≥12, ≥13, ≥14, ≥15, ≥16, ≥17,≥18, ≥19, ≥20, ≥30, ≥40, ≥50, ≥60, etc.) adjacent non-terminal residueslinked to other residues only by β-1,3 linkages. By “non-terminal” it ismeant that the residue is not present at a free end of the glucan. Insome embodiments, the adjacent non-terminal residues may not include anyresidues at which the cyclooctyne group is attached. The presence offive adjacent non-terminal residues linked to other residues only byβ-1,3 linkages may provide a protective antibody response, e.g. againstC. albicans.

In further embodiments, a conjugate may include two different glucanse.g. a first glucan having a β(1-3):β(1-6) ratio of between 1.5:1 and7.5:1, and a second glucan having exclusively or mainly β-1,3 linkages.For instance a conjugate may include both a laminarin glucan and acurdlan glucan.

Where a β-glucan includes both β-1,3 and β-1,6 linkages at a desiredratio and/or sequence then this glucan may be found in nature (e.g. alaminarin), or it may be made artificially. For instance, it may be madeby chemical synthesis, in whole or in part. Methods for the chemicalsynthesis of β-1,3/β-1,6 glucans are known, for example from references36-46. β-glucan including both β-1,3 and β-1,6 linkages at a desiredratio may also be made starting from an available glucan and treating itwith a β-1,6-glucanase (also known as glucan endo-1,6-β-glucosidase,1,6-β-D-glucan glucanohydrolase, etc.; EC 3.2.1.75) or a β-1,3-glucanase(such as an exo-1,3-glucanase (EC 3.2.1.58) or an endo-1,3-glucanase (EC3.2.1.39) until a desired ratio and/or sequence is reached.

When a glucan containing solely β-1,3-linked glucose is desired thenβ-1,6-glucanase treatment may be pursued to completion, asβ-1,6-glucanase will eventually yield pure β-1,3 glucan. Moreconveniently, however, a pure β-1,3-glucan may be used. These may bemade synthetically, by chemical and/or enzymatic synthesis e.g. using a(1→3)-β-D-glucan synthase, of which several are known from manyorganisms (including bacteria, yeasts, plants and fungi). Methods forthe chemical synthesis of β-1,3 glucans are known, for example fromreferences 47-50. As a useful alternative to synthesis, a naturalβ-1,3-glucan may be used, such as a curdlan (linear (β-1,3-glucan froman Agrobacterium previously known as Alcaligenes faecalis var.myxogenes; commercially available e.g. from Sigma-Aldrich catalog C7821)or paramylon (β-1,3-glucan from Euglena). Organisms producing highlevels of β-1,3-glucans are known in the art e.g. the Agrobacterium ofrefs. 51 & 52, or the Euglena gracilis of ref. 53.

Laminarin and curdlan are typically found in nature as high molecularweight polymers e.g. with a molecular weight of at least 100 kDa. Theyare often insoluble in aqueous media. In their natural forms, therefore,they are not well suited to immunisation. Thus the invention may use ashorter glucan e.g. those containing 60 or fewer glucose monosaccharideunits (e.g. 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45,44, 43, 42, 41, 40 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27,26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4). A glucan having a number of glucose residues in therange of 2-60 may be used e.g. between 10-50 or between 20-40 glucoseunits. A glucan with 25-30 glucose residues is particularly useful.Suitable glucans may be formed e.g. by acid hydrolysis of a naturalglucan, or by enzymatic digestion e.g. with a glucanase, such as aβ-1,3-glucanase. A glucan with 11-19, e.g. 13-19 and particularly 15 or17, glucose monosaccharide units is also useful. In particular, glucanswith the following structures (A) or (B) are specifically envisaged foruse in the present invention:

-   -   wherein n+2 is in the range of 2-60, e.g. between 10-50 or        between 2-40. Preferably, n+2 is in the range of 25-30 or 6-19,        e.g. 6 or 13-17. The inventors have found that n+2=6 is        suitable. n+2=15 may also be suitable

-   -   wherein n is in the range of 0-9, e.g. between 1-7 or between        2-6. Preferably, n is in the range of 3-4 or 1-3. The inventors        have found that n=2 is suitable.

In some embodiments, the glucan is a single molecular species. In theseembodiments, all of the glucan molecules are identical in terms ofsequence. Accordingly, all of the glucan molecules are identical interms of their structural properties, including molecular weight etc.Typically, this form of glucan is obtained by chemical synthesis, e.g.using the methods described above. For example, reference 48 describesthe synthesis of a single β-1,3 linked species. Alternatively, in otherembodiments, the glucan may be obtained from a natural glucan, e.g. aglucan from L. digitata, Agrobacterium or Euglena as described above,with the glucan being purified until the required single molecularspecies is obtained. Natural glucans that have been purified in this wayare commercially available. A glucan that is a single molecular speciesmay be identified by measuring the polydispersity (Mw/Mn) of the glucansample. This parameter can conveniently be measured by SEC-MALLS, forexample as described in reference 54. Suitable glucans for use in thisembodiment of the invention have a polydispersity of about 1, e.g. 1.01or less.

Solubility of natural glucans, such as curdlan, can be increased byintroducing ionic groups (e.g. by sulfation, particularly at 0-6 incurdlan). Such modifications may be used with the invention, but areideally avoided as they may alter the glucan's antigenicity.

When the saccharide is a glucan, it is typically a laminarin.

S. pneumoniae Capsular Saccharides

As discussed above, the saccharide may also be a bacterial capsularsaccharide. Further exemplary bacterial capsular saccharides includethose from S. pneumoniae. When the saccharide is a capsular saccharidesfrom S. pneumoniae, it is typically from one of the followingpneumococcal serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F, preferably from1, 5, 6B, 14, 19F and 23F. Capsular polysaccharides from S. pneumoniaecomprise repeating oligosaccharide units which may contain up to 8 sugarresidues. The oligosaccharide units for the main S. pneumoniae serotypesare described in refs 55 and 56.

S. aureus Capsular Saccharides

Further exemplary bacterial capsular saccharides include those from S.aureus, particularly the capsular polysaccharides of S. aureus type 5and type 8. The structures of type 5 and type 8 capsular polysaccharideswere described in references 57 and 58 as:

Type 5

-   -   →4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→        Type 8    -   →3)-β-D-ManNAcA(40Ac)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→Recent        NMR spectroscopy data [59] has led to a revision of these        structures to:        Type 5    -   →4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(30Ac)-(1→3)-β-D-FucNAc-(1→        Type 8    -   →3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

The polysaccharide may be chemically modified relative to the capsularpolysaccharide as found in nature.

For example, the polysaccharide may be de-O-acetylated (partially orfully), de-N-acetylated (partially or fully), N-propionated (partiallyor fully), etc. De-acetylation may occur before, during or afterconjugation, but typically occurs before conjugation. The effect ofde-acetylation etc. can be assessed by routine assays. For example, therelevance of O-acetylation on S. aureus type 5 or type 8 capsularpolysaccharides is discussed in reference 60. The native polysaccharidesare said in this document to have 75% O-acetylation. Thesepolysaccharides induced antibodies to both the polysaccharide backboneand O-acetyl groups. Polysaccharides with 0% O-acetylation stillelicited antibodies to the polysaccharide backbone. Both types ofantibody were opsonic against S. aureus strains that varied in theirO-acetyl content. Accordingly, the type 5 or type 8 capsularpolysaccharides used in the present invention may have between 0 and100% O-acetylation.

The degree of O-acetylation of the polysaccharide can be determined byany method known in the art, for example, by proton NMR (e.g. asdescribed in references 61, 62, 63 or 64). A further method is describedin reference 65. Similar methods may be used to determine the degree ofN-acetylation of the polysaccharide. O-acetyl groups may be removed byhydrolysis, for example by treatment with a base such as anhydroushydrazine [66] or NaOH [60]. Similar methods may be used to removeN-acetyl groups. To maintain high levels of O-acetylation on type 5and/or 8 capsular polysaccharides, treatments that lead to hydrolysis ofthe O-acetyl groups are minimised, e.g. treatments at extremes of pH.

Capsular polysaccharides can be purified by known techniques, asdescribed in the references herein. A typical process involvesphenol-ethanol inactivation of S. aureus cells, centrifugation,lysostaphin treatment, RNase/DNase treatment, centrifugation, dialysis,protease treatment, further dialysis, filtration, precipitation withethanol/CaCl₂, dialysis, freeze-drying, anion exchange chromatography,dialysis, freeze-drying, size exclusion chromatography, dialysis andfreeze-drying [67]. An alternative process involves autoclaving S.aureus cells, ultrafiltration of the polysaccharide-containingsupernatant, concentration, lyophilisation, treatment with sodiummetaperiodate to remove teichoic acid, further ultrafiltration,diafiltration, high performance size exclusion liquid chromatography,dialysis and freeze-drying [68].

The invention is not limited to polysaccharides purified from naturalsources, however, and the polysaccharides may be obtained by othermethods, such as total or partial synthesis.

Other Bacterial Capsular Saccharides

Further exemplary bacterial capsular saccharides include those fromHaemophilus influenzae Type b, Salmonella enterica Typhi Vi andClostridium difficile.

S. pyogenes (Group A Streptococcus or GAS) carbohydrate

The invention may also use non-capsular bacterial saccharides. Anexemplary non-capsular bacterial saccharides is the S. pyogenes GAScarbohydrate (also known as the GAS cell wall polysaccharide, or GASP).This saccharide features a branched structure with an L-rhamnopyranose(Rhap) backbone consisting of alternating alpha-(1→2) and alpha-(1→3)links and D-N-acetylglucosamine (GlcpNAc) residues beta-(1→3)-connectedto alternating rhamnose rings ([69]).

The GAS carbohydrate will generally be in its native form, but it mayhave been modified. For example, the saccharide may be shorter than thenative GAS carbohydrate, or may be chemically modified.

Thus the saccharide used according to the invention may be asubstantially full-length GAS carbohydrate, as found in nature, or itmay be shorter than the natural length. Full-length polysaccharides maybe depolymerised to give shorter fragments for use with the inventione.g. by hydrolysis in mild acid, by heating, by sizing chromatography,etc. A short fragment thought to correspond to the terminal unit on theGAS carbohydrate has been proposed for use in a vaccine [70].Accordingly, short fragments are envisaged in the present invention.However, it is preferred to use saccharides of substantiallyfull-length. The GAS carbohydrate typically has a molecular weight ofabout 10, in particular about 7.5-8.5 kDa. Molecular masses can bemeasured by HPLC, for example SEC-HPLC using a TSK Gel G3000SW column(Sigma) relative to pullulan standards, such as those available fromPolymer Standard Service [71].

The saccharide may be chemically modified relative to the GAScarbohydrate as found in nature. For example, the saccharide may bede-N-acetylated (partially or fully), N-propionated (partially orfully), etc. The effect of de-acetylation etc., for example onimmunogenicity, can be assessed by routine assays.

Derivatization

The present invention relates in part to a method of derivatizing asaccharide comprising attaching an eight-membered cycloalkyne group tothe saccharide.

The eight-membered cycloalkyne group is attached to the saccharide by acovalent linkage. Typically, the eight-membered cycloalkyne group isattached via a spacer. The eight-membered cycloalkyne group is typicallyat a terminus of the spacer. The other terminus of the spacer has afunctional group for attachment to the saccharide. The nature of thefunctional group will depend on the saccharide, in particular on thegroup or groups available on the saccharide for attachment. Attachmentof the eight-membered cycloalkyne group can be carried out using anysuitable method depending on the nature of the saccharide and, when aspacer is used, the functional group on the spacer.

For example, if the saccharide contains an amine, the spacer can includeany functional group that allows attachment to an amine (e.g. asuccinimidyl ester). Similarly, if the saccharide contains an aldehyde,the spacer can include any functional group that allows attachment to analdehyde (e.g. an amine).

In some embodiments, the eight-membered cycloalkyne group includes oneor more nitrogen atoms, such as 1, 2 or 3 nitrogen atoms. In someembodiments, the eight-membered cycloalkyne group is fused to one ormore other ring systems, such as cyclopropane or benzene. In onepreferred embodiment, the eight-membered cycloalkyne group is fused to acyclopropane group. In another preferred embodiment, the eight-memberedcycloalkyne group is fused to two benzene groups. In most preferredembodiments, the eight-membered cycloalkyne group is a cyclooctynegroup.

In one embodiment, the attachment is carried out using a compound havingthe formula X₁-L-X₂, where X₁ is the eight-membered cycloalkyne groupand X₂-L is the spacer. In these embodiments, X₂ may be any group thatcan react with a functional group on the saccharide, and L is a linkingmoiety in the spacer.

In some preferred embodiments, X₂ is N-oxysuccinimide. This group issuitable for attachment to an amine on a saccharide. In otherembodiments, X₂ may be an amine group, which is suitable for attachmentto an aldehyde on a saccharide. L may be a straight chain alkyl with 1to 10 carbon atoms (e.g. C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀) e.g.—(CH₂)₄— or —(CH₂)₃—. L typically has formula L³-L²-L¹-, in which L¹ iscarbonyl, L² is a straight chain alkyl with 1 to 10 carbon atoms (e.g.C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀) e.g. —(CH₂)₄— or —(CH₂)₅— or L²is absent, and L³ is NHC(O)—, carbonyl or —O(CH₃)—.

In one preferred embodiment, L¹ is carbonyl, L² is —(CH₂)₅— and L³ isNHC(O)—. In another preferred embodiment, L¹ is carbonyl, L² is —(CH₂)₄—and L³ is carbonyl. In another preferred embodiment, L¹ is carbonyl, L²is absent and L³ is —O(CH₃)—.

In one embodiment, X₁ is:

In another embodiment, X₁ is:

Preferably, X₁ is:

A preferred compound having the formula X₁-L-X₂ is:

Another preferred compound having the formula X₁-L-X₂ is:

A particularly preferred compound having the formula X₁-L-X₂ is:

Derivatization of the saccharide may be required to introduce functionalgroups such as amines and aldehydes. In some embodiments, attachment ofthe eight-membered cycloalkyne group to the saccharide is preceded byoxidation of the saccharide in order to introduce an aldehyde group intoat least one saccharide residue in the saccharide. This step may involvethe introduction of more than one aldehyde group into the saccharide.

For example, GBS capsular saccharides do not include an aldehyde groupin their natural form, and so it is typically generated beforeattachment of the cyclooctyne group by oxidation (e.g. periodateoxidation) of a portion (e.g. between 5 and 40%, particularly between 10and 30%, preferably about 20%) of the saccharide's sialic acid residues[72]. Alternatively, if the method uses a serotype V capsular saccharidethat is desialylated, then an aldehyde group may be generated in thissaccharide before attachment of the eight-membered cycloalkyne group byoxidation (e.g. periodate oxidation) of a portion (e.g. between 5 and40%, particularly between 10 and 30%, preferably about 20%) of thesaccharide's galactose residues [10].

Typical reactions to produce aldehydes include the use of periodatesalts, and particularly meta-periodates (e.g. sodium or potassiummeta-periodate e.g. NaIO₄), to oxidise hydroxyl groups [73]. The skilledperson would be capable of identifying suitable conditions foroxidation.

Oxidation of the saccharide may be followed by a step of reductiveamination, for example if it is desirable to provide an amine on thesaccharide for attachment to a spacer.

Reductive amination is a standard technique in organic chemistry. In oneembodiment, an aldehyde group in the saccharide residue reacts with anamine group in the spacer. This can conveniently be achieved bycombining the polysaccharide with the spacer in the presence of anappropriate reducing agent (e.g. cyanoborohydrides, such as sodiumcyanoborohydride NaBH₃CN; borane-pyridine; sodium triacetoxyborohydride;borohydride exchange resin; etc.). In another embodiment, an aldehydegroup is converted into an amine group by reductive amination to providean amine group for attachment of the spacer. The reductive aminationinvolves either ammonia or a primary amine (NH₂R). This can convenientlybe achieved by using an ammonium salt (e.g. ammonium chloride) incombination with an appropriate reducing agent (e.g. as listed above).The skilled person would be capable of identifying suitable conditionsfor reductive amination. For example, the inventors have found thattreatment of polysaccharide at 10 mg/ml with carrier protein at a 4:1polysaccharide:protein ratio (w/w) and NaBH₃CN at a 2:1polysaccharide:NaBH₃CN ratio is suitable.

When a spacer is used, the saccharide derivative will comprise a spacermoiety. The spacer moiety may include atoms such as carbon, hydrogen,oxygen and/or nitrogen. Spacers that comprise carbon and hydrogen aretypical, and spacers that further comprise oxygen and/or nitrogen arealso typically used. Spacers that include nitrogen atoms may include acarbon atom bonded to a nitrogen atom, which in turn is bonded to asecond carbon atom (—C—N—C—). Spacers that include an oxygen atomtypically include it as part of a carbonyl group. Spacer moieties with amolecular weight of between 30-500 Da are typical. Spacers containingtwo carbonyl groups are also typical.

A useful spacer moiety may be —NH—C(O)—(CH₂)_(n)—NH—C(O)—, wherein n is1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The value of n is typically 5. Theterminal —NH— in this spacer is usually attached to a carbon atom fromthe polysaccharide moiety. The terminal —C(O)— in this spacer is usuallyattached to the cyclooctyne group. A preferred spacer moiety canconveniently be introduced by a process involving: reductive aminationof the aldehyde in the oxidised saccharide residue; reaction of theresulting —NH₂ group with a bifunctional spacer that is a diester (e.g.a disuccinimidyl ester) of a dioic acid (e.g. of adipic acid,HOOC—(CH₂)₄—COOH); and reductive amination of the product ([74]).

Other chemistries that can be used to attach a spacer to a —NH₂ group inthe saccharide, include:

-   -   acryloylation (e.g. by reaction with acryloyl chloride),        followed by Michael-type addition to either the ϵ-NH₂ or to a        —SH [75]. The resulting spacer moiety is —NH—C(O)—(CH₂)₂—        (propionamido).    -   reaction with a haloacylhalide, followed by reaction with the        ϵ-NH₂ or to a —SH [76]. The spacer moiety is —NH—C(O)—CH₂—.

The method of derivatizing a saccharide according to the invention maygive the saccharide as described below.

The Saccharide Derivative

The invention provides a saccharide derivative comprising aneight-membered cycloalkyne group. The saccharide derivative may includeany saccharide and, where appropriate, spacer, as outlined above. Theinvention also provides a saccharide derivative obtained or obtainableby the method outlined above. The saccharide derivative is not anaturally occurring saccharide.

Preferred saccharide derivatives include a capsular saccharide fromStreptococcus agalactiae (“GBS”). In particularly preferred embodiments,the saccharide is a capsular saccharide from Streptococcus agalactiae(“GBS”) serotype II or V. In one embodiment, the saccharide derivativeis a GBS derivative having the following structure:

In another embodiment, the saccharide derivative is a GBS derivativehaving the following structure:

Conjugation Method

The invention relates in part to a method of conjugating a saccharidederivative as defined above to an azide-containing moiety, comprisingreacting the eight-membered cycloalkyne group with the azide to form atriazole linkage. In some embodiments, the saccharide derivative used inthe method of conjugation is produced according to the methods describedabove. In particular, the saccharide derivative may be produced byattaching an eight-membered cycloalkyne group to the saccharide. Themethod of conjugation is typically carried out in the absence of a metalcatalyst, such as a copper catalyst.

The inventors have found that a suitable conjugation method involvesmixing protein (typically at a concentration of 5 mg/ml) in phosphatebuffered saline (PBS), with saccharide (typically solubilized in waterat a concentration of about 25-30 mg/ml). Typically, the mixture ofprotein and saccharide will be stirred for about 6-12 hours at roomtemperature.

The method of conjugating a saccharide derivative to an azide-containingmoiety occurs via a [3+2] cycloaddition reaction. This reaction isfacilitated by the ring strain in the eight-membered cycloalkyne, whichpromotes the azide-alkyne cycloaddition reaction in the absence of acopper catalyst. The inventors have found that this method ofconjugation is particularly efficient, and is capable of producingconjugates in higher than were achievable using classical conjugationmethods. General methods for conjugation using a [3+2] cycloadditionreaction are known in the art and are disclosed in reference 77.

The method of conjugating a saccharide derivative to an azide-containingmoiety may give a conjugate as described below.

Azide-Containing Moiety

Typically, the azide-containing moiety is a carrier molecule, such as aprotein. The azide-containing moiety can be made according to methodsknown in the art, for example the methods disclosed in reference 78.

Useful carrier proteins include bacterial toxins or toxoids, such asdiphtheria toxoid or tetanus toxoid. Fragments of toxins or toxoids canalso be used e.g. fragment C of tetanus toxoid [79]. For example, theCRM197 mutant of diphtheria toxin [80-82] is a useful with theinvention. Other suitable carrier proteins include the N. meningitidisouter membrane protein [83], synthetic peptides [84,85], heat shockproteins [86,87], pertussis proteins [88,89], cytokines [90],lymphokines [90], hormones [90], growth factors [90], human serumalbumin (preferably recombinant), artificial proteins comprisingmultiple human CD4⁺ T cell epitopes from various pathogen-derivedantigens [91] such as N19 [92], protein D from H. influenzae [93,94],pneumococcal surface protein PspA [95], pneumolysin [96], iron-uptakeproteins [97], toxin A or B from C. difficile [98], recombinantPseudomonas aeruginosa exoprotein A (rEPA) [99], a GBS protein [100],etc. In preferred embodiments, the carrier protein is a GBS protein,such as GBS67 and GBS80 [101].

Typically, the azide-containing moiety includes a spacer. The azide istypically present as a terminal group in the azide-containing moiety,such that it is available to take part in the conjugation reactions asdescribed herein.

Spacers are used to attach an azide group to the moiety. Methods forattaching a spacer to a carrier molecule, such as a protein, are knownin the art (see e.g. reference 78).

The spacer may be a straight chain alkyl with 1 to 10 carbon atoms (e.g.C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀) e.g. —(CH₂)₄— or —(CH₂)₃—. Insome preferred embodiments, the spacer has the formula —[(CH₂)₂O]_(n)—,where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Suitably, n is 3.

When a spacer is used, the azide-containing moiety will comprise aspacer moiety. The spacer moiety may include atoms such as carbon,hydrogen, oxygen and/or nitrogen. Spacers that comprise carbon andhydrogen are typical, and spacers that further comprise oxygen and/ornitrogen are also typically used. Spacers that include nitrogen atomsmay include a carbon atom bonded to a nitrogen atom, which in turn isbonded to a second carbon atom (—C—N—C—). Spacers that include an oxygenatom typically include it as part of a carbonyl group. Spacer moietieswith a molecular weight of between 30-500 Da are typical. Spacerscontaining two carbonyl groups are also typical. A particularly usefulspacer moiety includes —[(CH₂)₂O]_(n)—, where n is 1, 2, 3, 4, 5, 6, 7,8, 9 or 10. Suitably, n is 3.

In preferred embodiments, the azide-containing moiety contains one ormore derivatized amino acids, such as one or more derivatized tyrosineresidues. Suitable methods for derivatizing tyrosine residues aredescribed in PCT/US2012/045549. In preferred embodiments, theazide-containing moiety is a carrier protein in which the azide isattached to the protein via a spacer. The azide-containing moiety may bea carrier protein in which the azide is attached to a derivatizedtyrosine residue on the protein via a spacer. The inventors have foundthat attaching the azide to a carrier protein via a tyrosine residue onthe protein is particularly preferred. In some embodiments, theazide-containing moiety is a carrier protein containing at least onederivatized tyrosine residue having the following structure, wherein theazide is attached via the 3H-1,2,4-triazole-3,5(4H)-dione:

For example, the azide-containing moiety may be a carrier proteincontaining at least one derivatized tyrosine residue having thefollowing structure:

The invention also provides azide-containing moieties as describedherein.

Conjugates

The invention relates in part to a conjugate of a saccharide derivativeas defined above and an azide-containing moiety as defined above,wherein the conjugate has the formula R—S-T, wherein R comprises aresidue of the saccharide derivative, S is a triazole group fused to aneight-membered cycloalkyl group and T comprises a residue of theazide-containing moiety.

In some embodiments, the eight-membered cycloalkyl group includes one ormore nitrogen atoms, such as 1, 2 or 3 nitrogen atoms. In someembodiments, the eight-membered cycloalkyl group is fused to one or moreother ring systems in addition to the triazole group, such ascyclopropane or benzene. In one preferred embodiment, the eight-memberedcycloalkyne group is fused to a cyclopropane group in addition to thetriazole group. In another preferred embodiment, the eight-memberedcycloalkyne group is fused to two benzene groups in addition to thetriazole group.

In a preferred embodiment, R—S-T is:

In another preferred embodiment, R—S-T is:

In a most preferred embodiment R—S-T is:

The moiety is typically a carrier molecule, such as a protein. Suitablecarrier proteins are described above. The conjugate may include a spacerin the residue of the saccharide derivative between the saccharide andS. For example, the spacer can be a spacer as described above for thesaccharide derivative. In addition or alternatively, the conjugate mayinclude a spacer in the residue of the azide-containing moiety betweenthe moiety and S. For example, the spacer can be a spacer as describedabove for the azide-containing moiety. Typically, the conjugate willinclude a spacer in the residue of the saccharide derivative between thesaccharide and S and a spacer in the residue of the azide-containingmoiety between the moiety and S.

In a particularly preferred embodiment, the conjugate includes GBSserotype V saccharide conjugated to GBS80 protein. In anotherparticularly preferred embodiment, the conjugate includes GBS serotypeII saccharide conjugated to GBS80 protein. In another particularlypreferred embodiment, the conjugate includes GBS serotype V saccharideconjugated to GBS67 protein. In another particularly preferredembodiment, the conjugate includes GBS serotype II saccharide conjugatedto GBS67 protein.

For example, the conjugate may have the following structure:

The conjugate may be obtained or obtainable by the method of conjugatinga saccharide derivative to an azide-containing moiety as describedabove.

In some embodiments, conjugates may have excess carrier protein (w/w) orexcess saccharide (w/w) e.g. in the ratio range of 1:5 to 5:1. Theconjugate may include small amounts of free (i.e. unconjugated) carrierprotein. When a given carrier protein is present in both free andconjugated form in a composition of the invention, the unconjugated formis preferably no more than 5% of the total amount of the carrier proteinin the composition as a whole, and more preferably present at less than2% (by weight). When the conjugate is comprised within a pharmaceuticalcomposition of the invention, the composition may also comprise freecarrier protein as immunogen [102]. After conjugation, free andconjugated antigens can be separated. There are many suitable methodse.g. hydrophobic chromatography, tangential ultrafiltration,diafiltration, etc. [see also refs. 103, 104 etc.].

Combinations of Conjugates and Other Antigens

As well as providing individual conjugates as described above, theinvention provides a composition comprising a conjugate of the inventionand one or more further antigens. The composition is typically animmunogenic composition.

The compositions of the invention may further comprise one or morefurther antigens, including additional bacterial, viral or parasiticantigens. These may be selected from the following:

-   -   a protein antigen from N. meningitidis serogroup B, such as        those in refs. 105 to 111, with protein ‘287’ (see below) and        derivatives (e.g. ‘ΔG287’) being particularly preferred.    -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 112, 113, 114, 115        etc.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 116 from        serogroup C or the oligosaccharides of ref. 117.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. refs.        118-120; chapters 22 & 23 of ref. 127].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 121, 122; chapter 15 of ref. 127].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 122,123; chapter 16 of ref. 127].    -   an antigen from hepatitis C virus [e.g. 124].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagluttinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 125 & 126; chapter 21 of ref.        127].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        13 of ref. 127].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 27 of        ref. 127].    -   a saccharide antigen from Haemophilus influenzae B [e.g. chapter        14 of ref. 127]    -   an antigen from N. gonorrhoeae [e.g. 105, 106, 107].    -   an antigen from Chlamydia pneumoniae [e.g. 128, 129, 130, 131,        132, 133, 134].    -   an antigen from Chlamydia trachomatis [e.g. 135].    -   an antigen from Porphyromonas gingivalis [e.g. 136].    -   polio antigen(s) [e.g. 137, 138; chapter 24 of ref. 127] such as        IPV.    -   rabies antigen(s) [e.g. 139] such as lyophilised inactivated        virus [e.g. 140, RabAvert™]    -   measles, mumps and/or rubella antigens [e.g. chapters 19, 20 and        26 of ref. 127].    -   influenza antigen(s) [e.g. chapters 17 & 18 of ref. 127], such        as the haemagluttinin and/or neuraminidase surface proteins.    -   an antigen from Moraxella catarrhalis [e.g. 141].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 142, 143, 144].    -   an antigen from Streptococcus agalactiae (group B streptococcus)        [e.g. 145-147].    -   an antigen from S. epidermidis [e.g. type I, II and/or III        saccharide obtainable from strains ATCC-31432, SE-360 and SE-10        as described in refs. 148, 149 and 150.

Where a saccharide or carbohydrate antigen is used, it is preferablyconjugated to a carrier in order to enhance immunogenicity. Conjugationof H. influenzae B, meningococcal and pneumococcal saccharide antigensis well known.

Toxic protein antigens may be detoxified where necessary (e.g.detoxification of pertussis toxin by chemical and/or genetic means[126]).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens.

Antigens may be adsorbed to an aluminium salt. Where there is more thanone conjugate in a composition, not all conjugates need to be adsorbed.

Antigens in the composition will typically be present at a concentrationof at least 1 μg/ml each. In general, the concentration of any givenantigen will be sufficient to elicit an immune response against thatantigen.

As an alternative to using proteins antigens in the composition of theinvention, nucleic acid encoding the antigen may be used [e.g. refs. 151to 159]. Protein components of the compositions of the invention maythus be replaced by nucleic acid (preferably DNA e.g. in the form of aplasmid) that encodes the protein. In practical terms, there may be anupper limit to the number of antigens included in compositions of theinvention. The number of antigens in a composition of the invention maybe less than 20, less than 19, less than 18, less than 17, less than 16,less than 15, less than 14, less than 13, less than 12, less than 11,less than 10, less than 9, less than 8, less than 7, less than 6, lessthan 5, less than 4, or less than 3. The number of antigens in acomposition of the invention may be less than 6, less than 5, or lessthan 4.

Pharmaceutical Compositions and Methods

The invention provides processes for preparing pharmaceuticalcompositions, comprising the steps of mixing conjugate of the inventionwith a pharmaceutically acceptable carrier. Typical ‘pharmaceuticallyacceptable carriers’ include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolisedmacromolecules such as proteins, saccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers,lactose, and lipid aggregates (such as oil droplets or liposomes). Suchcarriers are well known to those of ordinary skill in the art. Thevaccines may also contain diluents, such as water, saline, glycerol,etc. Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present. Sterilepyrogen-free, phosphate-buffered physiologic saline is a typicalcarrier. A thorough discussion of pharmaceutically acceptable excipientsis available in reference 160.

Compositions of the invention may be in aqueous form (i.e. solutions orsuspensions) or in a dried form (e.g. lyophilised). If a dried vaccineis used then it will be reconstituted into a liquid medium prior toinjection. Lyophilisation of conjugate vaccines is known in the art e.g.the Menjugate™ product is presented in lyophilised form, whereasNeisVac-C™ and Meningitec™ are presented in aqueous form. To stabiliseconjugates during lyophilisation, it may be typical to include a sugaralcohol (e.g. mannitol) or a disaccharide (e.g. sucrose or trehalose)e.g. at between 1 mg/ml and 30 mg/ml (e.g. about 25 mg/ml) in thecomposition.

The pharmaceutical compositions may be packaged into vials or intosyringes. The syringes may be supplied with or without needles. Asyringe will include a single dose of the composition, whereas a vialmay include a single dose or multiple doses.

Aqueous compositions of saccharides of the invention are suitable forreconstituting other vaccines from a lyophilised form. Where acomposition of the invention is to be used for such extemporaneousreconstitution, the invention provides a process for reconstituting sucha lyophilised vaccine, comprising the step of mixing the lyophilisedmaterial with an aqueous composition of the invention. The reconstitutedmaterial can be used for injection.

Compositions of the invention may be packaged in unit dose form or inmultiple dose form. For multiple dose forms, vials are preferred topre-filled syringes. Effective dosage volumes can be routinelyestablished, but a typical human dose of the composition has a volume of0.5 ml e.g. for intramuscular injection.

The pH of the composition is typically between 6 and 8, e.g. about 7.Stable pH may be maintained by the use of a buffer. If a compositioncomprises an aluminium hydroxide salt, it is typical to use a histidinebuffer [161]. The composition may be sterile and/or pyrogen-free.Compositions of the invention may be isotonic with respect to humans.

Compositions of the invention are immunogenic, and are more preferablyvaccine compositions. Vaccines according to the invention may either beprophylactic (i.e. to prevent infection) or therapeutic (i.e. to treatinfection), but will typically be prophylactic. Immunogenic compositionsused as vaccines comprise an immunologically effective amount ofantigen(s), as well as any other components, as needed. By‘immunologically effective amount’, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, age, the taxonomic group of individual to be treated (e.g.non-human primate, primate, etc.), the capacity of the individual'simmune system to synthesise antibodies, the degree of protectiondesired, the formulation of the vaccine, the treating doctor'sassessment of the medical situation, and other relevant factors. It isexpected that the amount will fall in a relatively broad range that canbe determined through routine trials.

Within each dose, the quantity of an individual saccharide antigen willgenerally be between 1-50 μg (measured as mass of saccharide) e.g. about1 μg, about 2.5 μg, about 4 μg, about 5 μg, or about 10 μg.

The compositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. The composition may be prepared forpulmonary administration e.g. as an inhaler, using a fine powder or aspray. The composition may be prepared as a suppository or pessary. Thecomposition may be prepared for nasal, aural or ocular administratione.g. as spray, drops, gel or powder [e.g. refs 162 & 163].

Success with nasal administration of pneumococcal saccharides [164,165],Hib saccharides [166], MenC saccharides [167], and mixtures of Hib andMenC saccharide conjugates [168] has been reported.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical.

Compositions of the invention will generally include a buffer. Aphosphate buffer is typical.

Compositions of the invention will generally be administered inconjunction with other immunoregulatory agents. In particular,compositions will usually include one or more adjuvants. Such adjuvantsinclude, but are not limited to:

Mineral-Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminium salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulphates, etc. [e.g. chapters 8 & 9 of ref. 169], or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being typical. Themineral containing compositions may also be formulated as a particle ofmetal salt [170].

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

A typical aluminium phosphate adjuvant is amorphous aluminiumhydroxyphosphate with PO₄/Al molar ratio between 0.84 and 0.92, includedat 0.6 mg Al³⁺/ml. Adsorption with a low dose of aluminium phosphate maybe used e.g. between 50 and 100 μg Al³⁺ per conjugate per dose. Where analuminium phosphate it used and it is desired not to adsorb an antigento the adjuvant, this is favoured by including free phosphate ions insolution (e.g. by the use of a phosphate buffer).

Oil Emulsions

Oil emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer) [Chapter 10 of ref. 169; also refs. 171-173]. MF59 isused as the adjuvant in the FLUAD™ influenza virus trivalent subunitvaccine.

Particularly useful adjuvants for use in the compositions are submicronoil-in-water emulsions. Preferred submicron oil-in-water emulsions foruse herein are squalene/water emulsions optionally containing varyingamounts of MTP-PE, such as a submicron oil-in-water emulsion containing4-5% w/v squalene, 0.25-1.0% w/v Tween 80 (polyoxyelthylenesorbitanmonooleate), and/or 0.25-1.0% Span 85 (sorbitan trioleate), and,optionally,N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphophoryloxy)-ethylamine(MTP-PE). Submicron oil-in-water emulsions, methods of making the sameand immunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in references 171 & 174-175.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

Saponin Formulations [Chapter 22 of Ref 169]

Saponin formulations may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia Saponaria Molina tree have been widely studied asadjuvants. Saponin can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla Paniculata (brides veil), and Saponariaofficinalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using HPLC and RP-HPLC. Specificpurified fractions using these techniques have been identified,including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C. Preferably, thesaponin is QS21. A method of production of QS21 is disclosed in ref.176. Saponin formulations may also comprise a sterol, such ascholesterol [177].

Combinations of saponins and cholesterols can be used to form uniqueparticles called immunostimulating complexes (ISCOMs) [chapter 23 ofref. 169]. ISCOMs typically also include a phospholipid such asphosphatidylethanolamine or phosphatidylcholine. Any known saponin canbe used in ISCOMs. Preferably, the ISCOM includes one or more of QuilA,QHA and QHC. ISCOMs are further described in refs. 177-179. Optionally,the ISCOMS may be devoid of additional detergent(s) [180].

A review of the development of saponin based adjuvants can be found inrefs. 181 & 182.

Virosomes and Virus-Like Particles

Virosomes and virus-like particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage, AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin refs. 183-188. Virosomes are discussed further in, for example, ref.189.

Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as non-toxic derivatives of enterobacterialliposaccharide (LPS), Lipid A derivatives, immunostimulatoryoligonucleotides and ADP-ribosylating toxins and detoxified derivativesthereof.

Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylatedmonophosphoryl lipid A with 4, 5 or 6 acylated chains. A preferred“small particle” form of 3 De-O-acylated monophosphoryl lipid A isdisclosed in ref. 190. Such “small particles” of 3dMPL are small enoughto be sterile filtered through a 0.22 μm membrane [190]. Other non-toxicLPS derivatives include monophosphoryl lipid A mimics, such asaminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [191,192].

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in refs. 193 & 194.

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (adinucleotide sequence containing an unmethylated cytosine linked by aphosphate bond to a guanosine). Double-stranded RNAs andoligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. References 195, 196 and 197 disclose possible analogsubstitutions e.g. replacement of guanosine with2′-deoxy-7-deazaguanosine. The adjuvant effect of CpG oligonucleotidesis further discussed in refs. 198-203.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT [204]. The CpG sequence may be specific for inducing a Th1 immuneresponse, such as a CpG-A ODN, or it may be more specific for inducing aB cell response, such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed inrefs. 205-207. Preferably, the CpG is a CpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers” (e.g.refs. 204 & 208-210).

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (E. coli heat labile enterotoxin “LT”), cholera(“CT”), or pertussis (“PT”). The use of detoxified ADP-ribosylatingtoxins as mucosal adjuvants is described in ref. 211 and as parenteraladjuvants in ref. 212. The toxin or toxoid is preferably in the form ofa holotoxin, comprising both A and B subunits. Preferably, the A subunitcontains a detoxifying mutation; preferably the B subunit is notmutated. Preferably, the adjuvant is a detoxified LT mutant such asLT-K63, LT-R72, and LT-G192. The use of ADP-ribosylating toxins anddetoxified derivatives thereof, particularly LT-K63 and LT-R72, asadjuvants can be found in refs. 213-220. Numerical reference for aminoacid substitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in ref. 221, specificallyincorporated herein by reference in its entirety.

Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5,IL-6, IL-7, IL-12 [222], etc.) [223], interferons (e.g. interferon-γ),macrophage colony stimulating factor, and tumor necrosis factor.

Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres [224] or mucoadhesives such as cross-linked derivatives ofpoly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone,polysaccharides and carboxymethylcellulose. Chitosan and derivativesthereof may also be used as adjuvants in the invention [225].

Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

Liposomes (Chapters 13 & 14 of Ref 169)

Examples of liposome formulations suitable for use as adjuvants aredescribed in refs. 226-228.

Polyoxyethylene Ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters [229]. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol [230] as well as polyoxyethylene alkyl ethers or estersurfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol [231]. Preferred polyoxyethylene ethersare selected from the following group: polyoxyethylene-9-lauryl ether(laureth 9), polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steorylether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether,and polyoxyethylene-23-lauryl ether.

Polyphosphazene (PCPP)

PCPP formulations are described, for example, in refs. 232 and 233.

Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), andN-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

Imidazoquinolone Compounds.

Examples of imidazoquinolone compounds suitable for use adjuvants in theinvention include Imiquamod and its homologues (e.g. “Resiquimod 3M”),described further in refs. 234 and 235.

Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in ref. 236.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

Tryptanthrin Compounds.

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in ref. 237. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the followingcombinations may be used as adjuvant compositions in the invention: (1)a saponin and an oil-in-water emulsion [238]; (2) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL) [239]; (3) a saponin (e.g.QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+a cholesterol; (4) asaponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol) [240]; (5)combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions [241]; (6) SAF, containing 10% squalane, 0.4% Tween 80™, 5%pluronic-block polymer L121, and thr-MDP, either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion. (7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing2% squalene, 0.2% Tween 80, and one or more bacterial cell wallcomponents from the group consisting of monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); and (8) one or more mineral salts (such as an aluminumsalt)+a non-toxic derivative of LPS (such as 3dMPL).

Other substances that act as immunostimulating agents are disclosed inchapter 7 of ref. 169.

The use of aluminium salt adjuvants is particularly useful, and antigensare generally adsorbed to such salts. The Menjugate™ and NeisVac™conjugates use a hydroxide adjuvant, whereas Meningitec™ uses aphosphate adjuvant. It is possible in compositions of the invention toadsorb some antigens to an aluminium hydroxide but to have otherantigens in association with an aluminium phosphate. Typically, however,only a single salt is used, e.g. a hydroxide or a phosphate, but notboth. Not all conjugates need to be adsorbed i.e. some or all can befree in solution.

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a pharmaceutical composition of theinvention to the mammal. The immune response is preferably protectiveand preferably involves antibodies. The method may raise a boosterresponse.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager; where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc. A preferred class of humans for treatment are patients at risk ofnosocomial infection, particularly those with end-stage renal diseaseand/or on haemodialysis. Other patients at risk of nosocomial infectionare also preferred, e.g. immunodeficient patients or those who haveundergone surgery, especially cardiac surgery, or trauma. Anotherpreferred class of humans for treatment are patients at risk ofbacteremia.

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine.

The invention also provides the use of a conjugate of the invention inthe manufacture of a medicament for raising an immune response in amammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by S. agalactiae e.g. neonatal sepsis orbacteremia, neonatal pneumonia, neonatal meningitis, endometritis,osteomyelitis, septic arthritis, etc.

The subject in which disease is prevented may not be the same as thesubject that receives the conjugate of the invention. For instance, aconjugate may be administered to a female (before or during pregnancy)in order to protect offspring (so-called ‘maternal immunisation’[242-244]).

One way of checking efficacy of therapeutic treatment involvesmonitoring GBS infection after administration of the composition of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses against the GBS antigens afteradministration of the composition.

Preferred compositions of the invention can confer an antibody titre ina patient that is superior to the criterion for seroprotection for eachantigenic component for an acceptable percentage of human subjects.Antigens with an associated antibody titre above which a host isconsidered to be seroconverted against the antigen are well known, andsuch titres are published by organisations such as WHO. Preferably morethan 80% of a statistically significant sample of subjects isseroconverted, more preferably more than 90%, still more preferably morethan 93% and most preferably 96-100%.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration. Intramuscular administration to the thigh or theupper arm is preferred. Injection may be via a needle (e.g. a hypodermicneedle), but needle-free injection may alternatively be used. A typicalintramuscular dose is 0.5 ml.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature (e.g., refs. 245-252,etc.).

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y. In some implementations, the term“comprising” refers to the inclusion of the indicated active agent, suchas recited polypeptides, as well as inclusion of other active agents,and pharmaceutically acceptable carriers, excipients, emollients,stabilizers, etc., as are known in the pharmaceutical industry. In someimplementations, the term “consisting essentially of” refers to acomposition, whose only active ingredient is the indicated activeingredient(s), however, other compounds may be included which are forstabilizing, preserving, etc. the formulation, but are not involveddirectly in the therapeutic effect of the indicated active ingredient.Use of the transitional phrase “consisting essentially” means that thescope of a claim is to be interpreted to encompass the specifiedmaterials or steps recited in the claim, and those that do notmaterially affect the basic and novel characteristic(s) of the claimedinvention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463(CCPA 1976); see also MPEP § 2111.03. Thus, the term “consistingessentially of” when used in a claim of this invention is not intendedto be interpreted to be equivalent to “comprising”.

The term “about” in relation to a numerical value x means, for example,x±10%.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Where the invention provides a process involving multiple sequentialsteps, the invention can also provide a process involving less than thetotal number of steps. The different steps can be performed at verydifferent times by different people in different places (e.g. indifferent countries).

It will be appreciated that sugar rings can exist in open and closedform and that, whilst closed forms are shown in structural formulaeherein, open forms are also encompassed by the invention. Similarly, itwill be appreciated that sugars can exist in pyranose and furanose formsand that, whilst pyranose forms are shown in structural formulae herein,furanose forms are also encompassed. Different anomeric forms of sugarsare also encompassed.

MODES FOR CARRYING OUT THE INVENTION

A. Saccharide Derivatization

GBS serotype II and V saccharides were reacted with NaIO₄ to effectoxidation of sialic acid residues to aldehyde groups. The extent ofoxidation of the sialyl moieties was controlled by varying the amount ofNaIO₄ used. Reductive amination of the aldehydes provided amine groupsfor the insertion of different spacers, facilitating attachment of thecyclooctyne group to the saccharides. Various cyclooctyne-containingcompounds were tested to establish the optimal length for the spacer, asshown in FIG. 1.

Reactions were monitored by NMR spectroscopy and carbohydrate recoverieswere quantified using colorimetric determination of sialic acid. FIG. 2shows a general reaction scheme for the attachment of a cyclooctynegroup to a GBS serotype II saccharide.

Using this method, two saccharide derivatives were produced, as shown inFIG. 3 (GBS serotype V saccharide with cyclooctyne group attached (I))and FIG. 4 (GBS serotype II saccharide with cyclooctyne group attached(II)).

The amount of each reactant used in the synthesis of GBS serotype Vsaccharide with cyclooctyne group attached (I) was as follows:

Compound MW mg mmol NH₂ eq ml GBS serotype V saccharide-NH₂ 1323 300.00453 Cyclooctyne-N-hydro- 380 13 10 xysuccinimide ester spacerTriethylamine 0.05 DMSO 3

The amount of each reactant used in the synthesis of GBS serotype IIsaccharide with cyclooctyne group attached (II) was as follows:

Compound MW mg mmol NH₂ eq ml GBS serotype II saccharide-NH₂ 1323 400.00393 Cyclooctyne-N-hydroxy- 380 13 10 succinimide ester spacerTriethylamine 0.04 DMSO 3

A further saccharide derivative was produced as shown in FIG. 10 (MenYsaccharide with cyclooctyne group attached (III)).

Saccharide derivatives were also synthesized using the other twocycloalkyne systems shown in FIG. 1.

B. Production and Purification of Conjugates

Spacers enabling conjugation of the protein (GBS60 or GBS67) wereinstalled by site directed Mannich-type reaction onto tyrosine using theprocedures described in PCT/US2012/045549, yielding carrier proteinattached to a terminal azide group. Carrier protein-azide was reactedwith the saccharide-cyclooctyne to effect an azide-alkyne cycloadditionreaction, yielding carrier protein-saccharide conjugate. FIG. 5 shows ageneral reaction scheme for the conjugation of saccharide derivative(II) to a GBS80 carrier protein via a tyrosine residue.

Eight different conjugates containing GBS protein were synthesised, asfollows (where “Y” denotes attachment to carrier protein GBS80 or GBS67via a tyrosine residue and “N₃” denotes the triazole linkage):

A. GBS80-Y—N₃-GBS serotype V saccharide

B. GBS80-Y—N₃-GB S serotype II saccharide

C. GBS67-Y—N₃-GBS serotype II saccharide

D. GBS67-Y—N₃-GBS serotype V saccharide

Conjugation was carried out at a saccharide:protein ratio of 6:1 (w/w)for conjugates A-B and at a saccharide:protein ration of 4:1 (w/w) forconjugates C-D. Addition of protein (in PBS) at a concentration of 5mg/ml to saccharide followed by stirring at room temperature for 6-12hours yielded conjugates. Conjugates were purified using ahydroxyapatite column to remove free protein (with a 2 mM NaPi, pH 7.2mobile phase buffer followed by a 400 mM NaPi, pH 7.2 mobile phasebuffer) and free saccharide (with a 2 mM NaPi, 550 mM NaCl, pH 7.2mobile phase buffer followed by a 10 mM NaPi, pH 7.2 mobile phase bufferfollowed by a 35 mM NaPi, pH 7.2 mobile phase buffer followed by a 400mM NaPi, pH 7.2 mobile phase buffer).

SDS-PAGE (3-8%) was used to confirm formation of the conjugates. Theresults of the SDS-PAGE characterization for each of conjugates A-D areshown in FIGS. 6-9, respectively.

HPAEC-PAD analysis was used to determine the saccharide content of theconjugates. The conjugates had the following properties:

Yield Free Total Saccharide/protein (% Saccharide/protein saccharideprotein used for final Conjugate Protein (w/w) (%) (mg) conjugation(w/w) protein) A GBS80 2.2 <5.5 648.0 6:1 21.1 B GBS80 2.7 <1.8 810.06:1 52.5 C GBS67 1.1 <4.5 975.0 4:1 29.5 D GBS67 2.5 <4.8 780.0 4:1 23.6

A conjugate containing CRM197 protein was also synthesised. Inparticular, saccharide derivative (III) was conjugated to CRM197protein, as follows:

E. CRM197-Y—N₃-MenY Saccharide

Conjugation was carried out using 60 equivalents of saccharidederivative (2.1 mg) and 1.5 mg of protein. SDS-PAGE was used to confirmformation of the conjugate. The results of the SDS-PAGE characterizationfor conjugate E are shown in FIG. 11.

C. Immunization Studies Using the Conjugates

The immunogenicity of various antigens was tested in mice as outlinedbelow.

Challenge Model Using Type V Strain

Groups of eight CD1 mice were immunised by intraperitoneal injectionwith a 1.0 μg dose of saccharide in an injection volume of 200 μl withAlumOH as adjuvant. Injections were carried out at 1, 21 and 35 days,with bleeding performed at 1, 35 and 49 days. Immunisations were carriedout in groups of eight mice with the following antigens: (i) PBS, (ii)CRM197-GBS serotype V saccharide, (iii) TT-GBS serotype V saccharide,(iv) GBS80-GBS serotype V saccharide and (v) GBS80-Y—N₃-GBS serotype Vsaccharide (conjugate A). Conjugates (i) to (iv) were prepared usingclassical conjugation methodologies (e.g. as disclosed in reference[253]), whereas conjugate (v) was prepared using click chemistry. Theneonates were challenged with type V strains. Results are shown below:

Antigen Protection/treated % Protection PBS 19/40 47 CRM197-GBS serotypeV 61/70 87 saccharide TT-GBS serotype V saccharide — — GBS80-GBSserotype V 54/57 95 saccharide GBS80-Y-N₃-GBS serotype V 23/70 33saccharide AChallenge Model Using Type II Strain

Groups of eight CD1 mice were immunised by intraperitoneal injectionwith a 1.0 μg dose of saccharide in an injection volume of 200 μl withAlumOH as adjuvant. Injections were carried out at 1, 21 and 35 days,with bleeding performed at 1, 35 and 49 days. Immunisations were carriedout in groups of eight mice with the following antigens: (i) PBS, (ii)CRM197-GBS serotype II saccharide, (iii) TT-GBS serotype II saccharide,(iv) GBS80-GBS serotype II saccharide and (v) GBS80-Y—N₃-GBS serotype IIsaccharide (conjugate B). Conjugates (i) to (iv) were prepared usingclassical conjugation methodologies, whereas conjugate (v) was preparedusing click chemistry. The neonates were challenged with type IIstrains. Results are shown below:

Antigen Protection/treated % Protection PBS 18/60 30 CRM197-GBS serotypeII 32/50 64 saccharide TT-GBS serotype II saccharide 19/30 63 GBS80-GBSserotype II 37/70 53 saccharide GBS80-Y-N₃-GBS serotype II 58/65 89saccharide B

These results show that higher levels of protection were achieved withGBS80-Y—N₃-GBS serotype II saccharide B than with the CRM197 and GBS80conjugates obtained using classical conjugation methods.

ELISA Immunoassay for Determining IgG Titers Against GBS Serotype IISaccharide Antigens

IgG titers against GBS serotype II saccharide in the sera from immunizedanimals were measured as follows. Microtiter plates were coated withantigens (e.g. GBS80-Y—N₃-GBS serotype II saccharide B) and the plateswere incubated overnight at room temperature and then washed three timesin washing buffer (0.05% Tween 20 in PBS). After dispensing 250 μl ofPBS, 2% BSA, 0.05% Tween 20 per well, plates were incubated 90 minutesat 37° C. and then aspirated to remove the post-coating solution. Testsera were diluted 1:400 in PBS, 2% BSA, 0.05% Tween 20. Standard serumwas prepared by pooling hyper immune sera and initial dilutions ofstandard pools were chosen to obtain an optical density (OD) of about2.000 at 405 nm. The plates were incubated for 1 hour at 37° C. and thenwashed with washing buffer and 100 μL of Alkaline Phosphatase-Conjugatedantimouse IgG 1:1000 in dilution buffer were dispensed in each well. Theplates were incubated 90 minutes at 37° C. and then washed with washingbuffer. 100 μL of a solution of p-NitroPhenylPhosphate (p-NPP) 4.0 mg/mLin substrate buffer were dispensed in each well. The plates wereincubated 30 minutes at room temperature and then 100 μL of a solutionof EDTA 7% (w/v) disodium salt plus Na₂HPO₄ 3.5% pH 8.0, were added toeach well to stop the enzymatic reaction. The optical density (OD) at405 nm was measured. Total IgG titres against GBS serotype II saccharideantigen were calculated by using the Reference Line Assay Method andresults were expressed as arbitrary ELISA Units/mL (EU/mL). For each ofthe three antigens, the standard serum IgG titer was arbitrarilyassigned a value of 1.0 EU/mL. The IgG titer of each serum was estimatedby interpolating the obtained ODs with the titration curve (bias andslope) of the standard pool.

The results are displayed in FIG. 12 (for 1.0 μg carbohydrate dose),FIG. 13 (for 0.5 μg carbohydrate dose) and FIG. 14 (for 1.0 μg proteindose). At 1.0 μg carbohydrate dose GBS80-Y—N₃-GBS serotype II saccharideB IgG titers are not statistically different from the CRM197 and GBS80conjugates obtained using classical conjugation methods. At 0.5 μgcarbohydrate dose GBS80-Y—N₃-GBS serotype II saccharide B IgG titers arenot statistically different from all the new conjugates and control.

Opsonophagocytosis Assay

The opsonophagocytosis assay was performed using GBS strains as targetcells and HL-60 cell line (ATCC; CCL-240), differentiated intogranulocyte-like cells, by adding 100 mM N,N dimethylformamide (Sigma)to the growth medium for 4 days. Mid-exponential bacterial cells wereincubated at 37° C. for 1 h in the presence of phagocytic cells, 10%baby rabbit complement (Cedarlane), and heat-inactivated mouse antisera.Negative controls consisted of reactions either with preimmune sera, orwithout HL-60, or with heat-inactivated complement. The amount ofopsonophagocytic killing was determined by subtracting the log of thenumber of colonies surviving the 1-h assay from the log of the number ofCFU at the zero time point. Results of the experiments are shown in FIG.15 and below:

Antigen OPKA titer PBS 10 CRM197-GBS serotype II saccharide 2306GBS80-GBS serotype II saccharide 2443 GBS80-Y-N₃-GBS serotype IIsaccharide B 1415

GBS80-Y—N₃-GBS serotype II saccharide B OPKA and IgG titers arestatistically comparable to the CRM197 and GBS80 conjugates obtainedusing classical conjugation methods. OPKA and IgG titers show goodcorrelation with % of survival in challenge animal model.

Immunogenicity of Conjugates Prepared at Different Saccharide:ProteinRatios

Immune response was assessed against GBS serotype II saccharide (with1.0 μg protein dose) with conjugates having different saccharide:proteinratios. The results are shown in FIG. 16 and below:

PS/protein Protection/treated ratio (challenge Antigen (w/w) strainDK21) % Protection PBS  6/59 10 GBS80, GBS serotype II 42/79 53saccharide GBS80-GBS serotype II 1.8 36/60 60 saccharide GBS80-Y-N₃-GBS2.7 32/80 40 serotype II saccharide GBS80-Y-N₃-GBS 1.1 68/69 98 serotypeII saccharide

Immune response was also assessed against GBS80 (with 1.0 μg proteindose) with conjugates having different saccharide:protein ratios. Theresults are shown in FIG. 17 and below:

PS/protein Protection/treated ratio (challenge Antigen (w/w) strainCOH1) % Protection PBS 28/80 35 GBS80 35/60 58 GBS80, GBS serotype 37/5074 II saccharide GBS80-GBS serotype II 1.8 30/60 50 saccharideGBS80-Y-N₃-GBS 2.7 36/80 45 serotype II saccharide GBS80-Y-N₃-GBS 1.149/70 70 serotype II saccharideAssessment of Presence of Anti-Linker Antibodies

A construct was prepared via tyrosine selective conjugation of a MenYdimer to CRM197 (FIG. 18). Low levels of antibodies directed to thelinker were found.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

EMBODIMENTS

The invention includes the following numbered embodiments:

1. A method of derivatizing a saccharide comprising attaching aneight-membered cycloalkyne group to the saccharide.

2. The method of embodiment 1, wherein the eight-membered cycloalkynegroup is fused to a cyclopropane group.

3. The method of embodiment 1, wherein the eight-membered cycloalkynegroup is fused to two benzene groups.

4. The method of embodiment 1, wherein the eight-membered cycloalkynegroup is a cyclooctyne group.

5. The method of any preceding embodiment, wherein the saccharide is acapsular saccharide.

6. The method of any preceding embodiment, wherein the saccharide is aGBS capsular saccharide.

7. The method of any preceding embodiment, wherein the saccharide is aGBS saccharide from serotype Ia, Ib, II, III or V.

8. The method of any preceding embodiment, wherein the saccharide is aGBS saccharide from serotype II or V.

9. The method of any preceding embodiment, wherein the eight-memberedcycloalkyne group is attached to the saccharide via a spacer.

10. The method of embodiment 9, wherein the eight-membered cycloalkynegroup is on a terminus of the spacer.

11. The method of embodiment 10, wherein the other terminus of thespacer has a functional group for attachment to the saccharide.

12. The method of embodiment 11, wherein the attachment is carried outusing a compound having the formula X₁-L-X₂, where X₁ is theeight-membered cycloalkyne group and X₂-L is the spacer in which X₂ isany group that can react with a functional group on the saccharide and Lis a linking moiety in the spacer.

13. The method of embodiment 12, wherein X₂ is N-oxysuccinimide.

14. The method of embodiment 12 or 13, wherein L has the formula-L³-L²-L¹-, wherein L¹ is carbonyl, L² is a straight chain alkyl with 1to 10 carbon atoms or L² is absent, and L³ is —NHC(O)—, carbonyl or—O(CH₃)—.

15. The method of any one of embodiments 12 to 14, wherein the compoundhaving the formula X₁-L-X₂ is:

16. The method of any one of embodiments 12 to 14, wherein the compoundhaving the formula X₁-L-X₂ is:

17. The method of any one of embodiments 12 to 14, wherein the compoundhaving the formula X₁-L-X₂ is:

18. A saccharide derivative comprising an eight-membered cycloalkynegroup.

19. The saccharide derivative of embodiment 18, wherein theeight-membered cycloalkyne group is a cyclooctyne group.

20. The saccharide derivative according to embodiment 18, obtainable bythe method of any one of embodiments 1 to 17.

21. A method of conjugating a saccharide derivative as defined in anyone of embodiments 18 to 20 to an azide-containing moiety, comprisingreacting the eight-membered cycloalkyne group with the azide to form atriazole linkage.

22. The method of embodiment 21, wherein the saccharide derivative isproduced according to the method of any one of embodiments 1 to 17.

23. The method of embodiment 21 or embodiment 22, wherein the method iscarried out in the absence of a metal catalyst.

24. The method of any one of embodiments 21 to 23, wherein theconjugation occurs via a [3+2] cycloaddition reaction.

25. The method of any one of embodiments 21 to 25, wherein theazide-containing moiety is a carrier molecule.

26. The method of embodiment 25, wherein the carrier molecule is aprotein.

27. The method of embodiment 26, wherein the protein is a GBS protein.

28. The method of embodiment 27, wherein the GBS protein is GBS67 orGBS80.

29. The method of any one of embodiments 21 to 28, wherein theazide-containing moiety includes a spacer.

30. The method of embodiment 29, wherein the azide-containing moiety isa carrier protein containing at least one derivatized tyrosine residuehaving the following structure, wherein the azide is attached via the3H-1,2,4-triazole-3,5(4H)-dione:

31. The method of any one of embodiments 21 to 30, wherein the azide ispresent as a terminal group in the azide-containing moiety.

32. The method of embodiment 31, wherein the azide-containing moiety isa carrier protein containing at least one derivatized tyrosine residuehaving the following structure:

33. A conjugate of a saccharide derivative as defined in any one ofembodiments 18 to 20 and an azide-containing moiety, wherein theconjugate has the formula R—S-T, wherein R comprises a residue of thesaccharide derivative, S is a triazole group fused to an eight-memberedcycloalkyl group and T comprises a residue of the azide-containingmoiety.

34. The conjugate of embodiment 33, wherein the conjugate includes aspacer in the residue of the saccharide derivative between thesaccharide and S.

35. The conjugate of embodiment 34, wherein the spacer has the formula—NH—C(O)—(CH₂)_(n)—NH—C(O)—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

36. The conjugate of embodiment 35, wherein n is 5.

37. The conjugate of any one of embodiments 33 to 36, wherein theconjugate includes a spacer in the residue of the azide-containingmoiety between the moiety and S.

38. The conjugate of embodiment 37, wherein the spacer has the formula—[(CH₂)₂O]_(n)—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

39. The conjugate of embodiment 38, wherein n is 3.

40. The conjugate of any one of embodiments 33 to 39, wherein theconjugate includes a spacer in the residue of the saccharide derivativebetween the saccharide and S and a spacer in the residue of theazide-containing moiety between the moiety and S.

41. The conjugate of any one of embodiments 33 to 40, wherein R—S-T is:

42. The conjugate of any one of embodiments 33 to 40, wherein R—S-T is:

43. The conjugate of any one of embodiments 33 to 40, wherein R—S-T is:

44. The conjugate of any one of embodiments 33 to 43, obtainable by themethod of any one of embodiments 21 to 32.

45. A pharmaceutical composition comprising a conjugate of the inventionin combination with a pharmaceutically acceptable carrier.

46. A method for raising an immune response in a mammal, comprisingadministering a conjugate or pharmaceutical composition according to anyone of embodiments 33 to 43 to the mammal.

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The invention claimed is:
 1. A compound comprising (a) a saccharide and(b) an eight-membered cyclooctyne group, wherein the eight-memberedcyclooctyne group is

wherein (a) and (b) are covalently linked via a spacer having theformula —NH—C(O)—(CH₂)—NH—C(O)—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10; and wherein the terminal —NH— is attached to a carbon atom of thesaccharide and the terminal —C(O)— is attached to the eight-memberedcyclooctyne group.
 2. The compound of claim 1, obtained by a method ofderivatizing comprising attaching the eight-membered cyclooctyne groupto the saccharide, wherein the saccharide is a capsular saccharide.
 3. Aconjugate formed from (1) a compound comprising a saccharide covalentlylinked via a spacer to a cyclooctyne group and (2) a protein comprisingan azide moiety, wherein the conjugate is formed by reacting thecyclooctyne group with the azide moiety to form a triazole linkage,wherein the spacer has the formula —NH—C(O)—(CH₂)—NH—C(O)—, where n is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and wherein the terminal —NH— isattached to a carbon atom of the saccharide and the terminal —C(O)— isattached to the cyclooctyne group.
 4. The conjugate of claim 3, whereinn is
 5. 5. The conjugate of claim 3, wherein the protein is GBS80. 6.The conjugate of claim 3, wherein the saccharide is a GBS serotype IIsaccharide.
 7. The conjugate of claim 5, wherein the saccharide is a GBSserotype II saccharide.
 8. A pharmaceutical composition comprising aconjugate of claim 3 in combination with a pharmaceutically acceptablecarrier.